This is a broad project aimed at the understanding of the details of the interaction between protein inhibitors and the proteinases they inhibit. Particular stress is laid upon determining the specificity of the interaction and the role of the individual amino acid residues in the reactive sites of the inhibitors. The approach is three pronged. It consists of 1) in depth physicochemical studies on the detailed mechanism of the enzyme-inhibitor interaction. An attempt is being made to determine all of the equilibrium constants and rate constants involved in the interaction over broad ranges of pH and of solvent composition, 2) screening of the specificity of various enzyme-inhibitor pairs. When interesting homologous pairs where one inhibitor inhibits a particular enzyme and the other does not amino acid sequences of the reactive sites of the two inhibitors are determined. Total sequencing work is also going on on avian ovomucoids, 3) The second subproject results in hypotheses which amino acid residue change in a mutant pair is responsible for the change in inhibitory specificity. To test the validity of such hypotheses the residue in question is altered in the reactive site of soybean trypsin inhibitor (Kunitz) by an appropriate semisynthetic modification. Strategies have now been developed to alter either of the two residues immediately adjacent to the reactive site (P1 and P1'). Work is in progress to alter the penultimate residues P2 and P2'.